Pseudogenes generate endogenous small interfering RNAs (endo-siRNAs) in mouse oocytes, which regulate gene expression. These endo-siRNAs are derived from double-stranded RNAs formed by spliced transcripts from protein-coding genes and antisense transcripts from homologous pseudogenes, or from inverted repeat pseudogenes. They may also repress mobile genetic elements along with Piwi-interacting RNAs (piRNAs). Loss of Dicer, essential for small RNA production, increases expression of endo-siRNA targets, showing their regulatory role. Pseudogenes thus contribute to gene regulation via the RNA interference pathway, possibly explaining the evolutionary pressure to conserve argonaute-mediated catalysis in mammals. Small RNA pathways in mammals include miRNAs and piRNAs, which target different downstream elements. piRNAs are found in germ cells and some gonadal somatic cells, and are involved in repressing transposable elements. In mice, mutations in Piwi family members cause male sterility and gonadal hypotrophy, while female germ cells remain normal. Small RNA profiles in oocytes revealed a piRNA population similar to that in spermatocytes, with many sequences matching annotated repeats and some unannotated regions. These piRNAs arise from discrete genomic loci, often with structural similarities to Drosophila piRNA loci, and are involved in transposon repression. siRNAs and piRNAs both regulate transposons in oocytes. siRNAs are derived from transposon transcripts and may be generated from inverted repeats. piRNAs are produced from genomic loci and may act in a feed-forward loop with transposon transcripts. Both siRNAs and piRNAs are involved in transposon repression, with siRNAs being more abundant but piRNA cloning frequencies reduced by 2'-O-methyl modification. The study also shows that pseudogene-derived antisense siRNAs can regulate protein-coding genes through RNA interference. Dicer loss increases expression of endo-siRNA targets, indicating their regulatory role. Pseudogenes are often diverged from their functional ancestors but can generate antisense siRNAs that regulate homologous genes. The study shows that pseudogene-derived siRNAs can target genes like Hdac1, directing RISC to cleave mRNAs. The conservation of argonaute activity in mammals is supported by the role of endogenous siRNAs in gene regulation. The unique environment of oocytes, lacking a protein kinase R response, allows for the production of dsRNAs from sense and antisense transcripts, which are processed into siRNAs. These siRNAs may regulate microtubule dynamics and other processes essential for oocyte development. The findings highlight the role of pseudogenes in generating functional siRNAs and their involvement in gene regulation in mammalian oocytes.Pseudogenes generate endogenous small interfering RNAs (endo-siRNAs) in mouse oocytes, which regulate gene expression. These endo-siRNAs are derived from double-stranded RNAs formed by spliced transcripts from protein-coding genes and antisense transcripts from homologous pseudogenes, or from inverted repeat pseudogenes. They may also repress mobile genetic elements along with Piwi-interacting RNAs (piRNAs). Loss of Dicer, essential for small RNA production, increases expression of endo-siRNA targets, showing their regulatory role. Pseudogenes thus contribute to gene regulation via the RNA interference pathway, possibly explaining the evolutionary pressure to conserve argonaute-mediated catalysis in mammals. Small RNA pathways in mammals include miRNAs and piRNAs, which target different downstream elements. piRNAs are found in germ cells and some gonadal somatic cells, and are involved in repressing transposable elements. In mice, mutations in Piwi family members cause male sterility and gonadal hypotrophy, while female germ cells remain normal. Small RNA profiles in oocytes revealed a piRNA population similar to that in spermatocytes, with many sequences matching annotated repeats and some unannotated regions. These piRNAs arise from discrete genomic loci, often with structural similarities to Drosophila piRNA loci, and are involved in transposon repression. siRNAs and piRNAs both regulate transposons in oocytes. siRNAs are derived from transposon transcripts and may be generated from inverted repeats. piRNAs are produced from genomic loci and may act in a feed-forward loop with transposon transcripts. Both siRNAs and piRNAs are involved in transposon repression, with siRNAs being more abundant but piRNA cloning frequencies reduced by 2'-O-methyl modification. The study also shows that pseudogene-derived antisense siRNAs can regulate protein-coding genes through RNA interference. Dicer loss increases expression of endo-siRNA targets, indicating their regulatory role. Pseudogenes are often diverged from their functional ancestors but can generate antisense siRNAs that regulate homologous genes. The study shows that pseudogene-derived siRNAs can target genes like Hdac1, directing RISC to cleave mRNAs. The conservation of argonaute activity in mammals is supported by the role of endogenous siRNAs in gene regulation. The unique environment of oocytes, lacking a protein kinase R response, allows for the production of dsRNAs from sense and antisense transcripts, which are processed into siRNAs. These siRNAs may regulate microtubule dynamics and other processes essential for oocyte development. The findings highlight the role of pseudogenes in generating functional siRNAs and their involvement in gene regulation in mammalian oocytes.